PROJECT SUMMARY Cholera is a severe dehydrating disease caused by Vibrio cholerae. Unfortunately, we do not yet know what is the prime functional mediator of protection against cholera. This deficit has impacted development of cholera vaccines, with currently available cholera vaccines providing lower-level and shorter-term protection against cholera, especially in children, than that afforded by previous wild type disease. We and others have generated strong preliminary evidence that protective immunity against cholera is mediated by antibodies at the mucosal surface, and that V. cholerae O-specific polysaccharide (OSP) is the primary antigenic target. V. cholerae is a motile non-invasive enteropathogen with a single polar sheathed flagellum (covered by LPS/OSP). We hypothesize that differences in the follicular T cell immune response between vaccinees and infected cholera patients suppress the immune responses in vaccinees. In addition, we also hypothesize that these differences affect the development, maturation, and functionality of antibody responses against key Vibrio cholerae antigens, O-specific polysaccharide (OSP) and cholera toxin (CT). To address these hypotheses, we propose three specific aims. Aim #1: We will use flow cytometry of mononuclear cells isolated from patients with cholera to evaluate follicular T cells (TFH) in relation to infection at different days after onset of disease and following vaccination. Aim #2: We will use a novel nano- well approach to isolate single cholera antigen-specific cells by phenotypic characterization in duodenal biopsy specimens (and peripheral blood) of individuals recovering from cholera. We will clone the immunoglobulin chains from these recovered lymphocytes, including plasmablasts, plasma cells, and memory B cells, and we will evaluate cloned antibodies for functionality, as well as affinity maturation and somatic hyper-mutation; we will perform a similar analysis of blood samples of vaccinees. Aim #3: We will assess for the presence of V. cholerae agglutinating and anti-motility antibodies in patients with cholera, as well as in vaccinees. This work builds upon a highly-successful and ongoing international collaborative effort between researchers at the International Centre for Diarrhoeal Disease Research in Dhaka, Bangladesh (icddr,b), the Massachusetts General Hospital- Harvard in Boston and Emory University, Atlanta, USA. The goal of this K43 Emerging Global Leader Career Development Program would be to strengthen the skills necessary to become an independent researcher/investigator in mucosal immunology in resource-limited settings.